The goal of this research project has been and remains to define the mechanism responsible for the resistance of tumor cells with abnormal expression of the Bcl-2 oncogene to cancer chemotherapy agents. Bcl-2 is the prototypic member of a family of proteins, whose main function appears to be the regulation of apoptosis. Bcl-2 is highly expressed in a variety of human cancers and provides a growth advantage to tumors by blocking apoptosis during tumor progression. However, this block also renders such tumor cells resistant to anticancer therapies including most cancer chemotherapy drugs that rely on apoptosis as their primary mode of cell killing. Indeed, Bcl-2 expression produces a multi-drug resistance (MDR) phenotype. The mechanism by which Bcl-2 regulates apoptosis is not understood. We have previously shown that Bcl-2 expressing cells have higher intracellular levels of glutathione (GSH). As we demonstrate in this application, the mitochondria from Bcl-2 expressing cells also have higher GSH levels and have enhanced capacity to actively transport GSH from the cytosol into mitochondria. These are novel findings that are entirely consistent with the ability of Bcl-2 to suppress mitochondrial apoptosis; it is our contention that MDR due to Bcl-2 expression could be reversed by inhibiting or reversing Bcl-2's enhancement of mitochondrial GSH levels. Therefore, in this application, Specific Aims 1 and 2 are directed at characterizing this effect of Bcl-2 on mitochondrial GSH transport and testing the hypothesis that the level of mitochondrial GSH directly regulates mitochondrial apoptosis. In Aim 3, we propose to follow up on a new lead and investigate whether Bcl-2 protein is redistributed to the plasma membrane after cells receive an apoptotic signal. Finally, we propose in Specific Aim 4 to investigate strategies for overcoming Bcl-2 mediated MDR through the use of drugs that enable GSH efflux from cells or mitochondria. The significance of this project relates to the problem of MDR in cancer therapeutics. It is our central hypothesis that understanding the biochemical and molecular basis for Bcl-2's ability to block anticancer-drug induced apoptosis will lead to the development of strategies for reversing this drug resistance and enhancing therapeutic effects.